Tolerance to injected opiates can be demonstrated in an in vitro system, namely, the electrically stimulated guinea pig ileum. A very large dose of morphine (750 milligrams/kg) is injected into a naive guinea pig causing a complete block of contractions elicited by electrical stimulation of the longitudinal muscle removed 1 1/2 hours later. This same dose is almost completely ineffective when injected into guinea pigs administered morphine, 100 milligrams/kg/day for 10 days, i.e., tissue tolerance has developed to the challenging dose of the drug. We intend to compare a large number of properties of the ileum from normal and chronically morphinized guinea pigs in order to gain insight into the mechanism(s) involved in development of tolerance. Such studies will be carried out primarily using naptosomes prepared from this tissue since most likely this is the precise locus of tolerance development. The following properties of such synaptosomes will be examined: rate of metabolism of morphine, number of opiate receptors present, levels of morphine and metabolites following injection, protein and nucleic acid metabolism, and activity of cholinesterase and choline acetyltransferase. In addition, we will examine the effect of inhibitors of protein and nucleic acid synthesis on the time course and degree of development of tolerance as well as the possible involvement of the adenylate cyclase system in this phenomenon.